Targeted gas-filled microvesicles

ABSTRACT

Gas-filled microvesicles associated with a polypeptide comprising a sequence of amino acids, said sequence exhibiting binding affinity for selectins, particularly p-selectin. The gas-filled microvesicles can be used in ultrasound imaging.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the national stage application of correspondinginternational application number PCT/EP2011/063720 filed Aug. 9, 2011,which claims priority to and the benefit of European application no.EP10172318.7, filed Aug. 9, 2010, all of which are hereby incorporatedby reference in their entirety.

The instant application contains a Sequence Listing which is beingsubmitted in compliance with the code set forth in the tables in WIPOStandard ST.25 (1998), Appendix 2 via EFS-WEB and is hereby incorporatedby reference in its entirety.

TECHNICAL FIELD

The invention relates in general terms to targeted gas-filledmicrovesicles and to aqueous suspensions containing said microvesicles,for use in particular in diagnostic methods.

BACKGROUND OF THE INVENTION

Rapid development of contrast agents in the recent years has generated anumber of different formulations, which are useful in contrast-enhancedimaging of organs and tissue of human or animal body.

More recently, attention has been given to so-called “molecularimaging”, where suitable target specific components are used in theformulation of the contrast agents, for allowing selectivecontrast-enhanced imaging of organs or tissues. In addition, therapeuticuse of contrast agent formulations, optionally in combination withmolecular imaging, has also been described.

A class of contrast agents, particularly useful for ultrasound contrastimaging, includes suspensions of gas bubbles of nano- and/ormicro-metric size dispersed in an aqueous medium. Of particular interestare those formulations where the gas bubbles are stabilized, for exampleby using emulsifiers, oils, thickeners or sugars, or by entrapping orencapsulating the gas or a precursor thereof in a variety of systems.These stabilized gas bubbles are generally referred to in the art withvarious terminologies, depending typically from the stabilizing materialemployed for their preparation; these terms include, for instance,“microspheres”, “microbubbles”, “microcapsules” or “microballoons”. Theterm “gas-filled microvesicles”, or shortly “microvesicles”, as usedherein includes any of the above terminology.

The formulations of gas-filled microvesicles can be suitably modified,either for improving the diagnostic effect (e.g. through molecularimaging) and/or for therapeutic purposes, such as drug delivery and/orthrombolysis. For instance, microvesicles can be associated (e.g. byinclusion in their boundary envelope) with therapeutic agents and/orwith specific components which are capable to link to a determinedtarget within a patient's body (known as “targeting ligands”). Examplesof targeting ligands include, for instance, peptides, proteins,antibodies, aptamers or carbohydrates capable of binding to specificreceptors expressed by organs or tissues during pathogenic processessuch as, for instance, angiogenesis, inflammation or thrombus formation.

Selectins (in particular P-, L- and E-selectin) are cell adhesionmolecules expressed, among others, by vascular endothelium duringinflammation processes. Selectin ligands and, in particular, P-selectinglycoprotein ligand-1 (PSGL-1: GenBank Acc. No Q14242.1), is expressedconstitutively on all leukocytes (neutrophiles, monocytes and mostlymphocytes) and myeloid cells. As such, it plays a critical role in thetethering of these cells to activated platelets or endothelia expressingP-selectin and, even though with a lower affinity, to E and L-selectin.Examples of P-Selectin ligands are disclosed for instance in U.S. Pat.No. 5,840,679

International Patent Application Publ. No. WO 2008/131217 disclosesmicrobubble compositions comprising targeting ligands directed toP-selectin. In particular, the targeting ligand is a fusion proteincomprising a P-selectin ligand and a dimerization domain. In practicalembodiments, the Application discloses the use of recombinant P-selectinligand composed of the amino terminal region of PSGL-1 in aselectin-binding glycoform fused to the Fc portion of human IgG₁(rPSGL-Ig), to be conjugated via biotin-streptavidin binding to biotincontaining microbubbles. While said Application does not disclose anyexact sequence of the P-Selecting ligand, it refers to examples ofP-selectin ligands and fragments thereof disclosed by US PatentApplication Publ. No. 2003/0166521.

US 2003/0166521 discloses a PSGL-1 fusion protein (dimPSGL-1), alsoreferred to as recombinant PSGL-Ig (or rPSGL-Ig), produced by truncatingthe N-term 47 amino acids of mature PSGL-1 and linking said N-term 47amino acids of PSGL-1 to a Fc portion of human immunoglobulin G-1(IgG₁).

The Applicant has now observed that microvesicles bearing only afragment of said rPSGL-1 protein may have some advantages when comparedwith microvesicles bearing the complete protein, for instance in termsof binding efficacy and/or in terms of stability of an aqueoussuspension containing the microvesicles.

SUMMARY OF THE INVENTION

An aspect of the invention relates to an aqueous suspension ofgas-filled microvesicles associated with a polypeptide consisting of asequence of at most 200 amino acid residues and comprising at leastamino acids 5-16 as set forth in SEQ ID NO:1.

Preferably said polypeptide is associated with a component of themicrovesicle, more preferably through a covalent bond.

Preferably, said polypeptide comprises at least amino acid 1-19 as setforth in SEQ ID NO: 1, more preferably at least amino acid 5-41 as setforth in SEQ ID NO: 1, and even more preferably at least amino acid 1-47as set forth in SEQ ID NO: 1.

According to a preferred embodiment, said polypeptide comprises at most100 amino acid residues, more preferably at most 75 amino acid residues.

According to a preferred embodiment, said polypeptide consists of theamino acid sequence of formula (I):(X^(A))_(n)—Y—(X^(B))_(m)  (I)wherein:

(X^(A)), represents a sequence of n amino acids X^(A), comprising atleast amino acids 5-16 as set forth in SEQ ID NO:1, where:

-   -   n is an integer of from 12 to 199; and    -   X^(A) represents any amino acid with the exception of lysine;

(X^(B))_(m) represents a sequence of m amino acids X^(B), where;

-   -   m is an integer of from 0 to 10, with the proviso that the sum        m+n is at most 199; and    -   X^(B) represents any amino acid with the exception of lysine and        cysteine; and

Y represents an amino acid comprising a reactive moiety for associatingthe polypeptide with a component of the microvesicle.

Preferably the reactive moiety of the Y residue is —NH₂ or —SH; morepreferably Y is lysine or cysteine, even more preferably lysine.

According to a further preferred embodiment, said polypeptide isrepresented by a sequence of formula (II):(X¹)_(p)—[(C—(X²)_(a)]_(q)—[(C—(X³)_(b)]_(r)—Y—(X⁴)_(s)  (II)

wherein

C represents Cysteine;

Y represents Lysine or Cysteine, preferably Lysine;

(X¹)_(p) represents a sequence of p amino acids X¹ comprising at leastamino acids 5-16 as set forth in SEQ ID NO:1, (X²), represents asequence of a amino acids X², (X³)_(b) represents a sequence of b aminoacids X³, (X⁴)_(s) represents a sequence of s amino acids X², where:

-   -   X¹, X², X³, X⁴ independently represent any amino acid with the        exception of lysine and cysteine;    -   p is an integer of from 12 to 199, preferably from 12 to 99,        more preferably 12 to 74;    -   a and b are independently an integer of from 0 to 50, preferably        0 to 20 and more preferably from 0 to 10;    -   q and r are independently 0 or 1, at least one being 1; and    -   s is an integer of from 0 to 10;    -   with the proviso that the sum p+(a·q)+(b·r)+s is at most 199,        preferably at most 99 and even more preferably 74,

The above illustrated polypeptides preferably comprise at least oneO-glycan moiety and/or at least one sulfate residue bound to an aminoacid of the sequence.

According to a preferred embodiment of the invention, the aboveillustrated polypeptide is in dimeric form, preferably in homodimericform.

According to a particularly preferred embodiment, said polypeptideconsists of a sequence as set forth in SEQ ID NO:3. Preferably, the twocysteine residues of the sequence may bind to respective cysteineresidues of a corresponding sequence, to provide the polypeptide indimeric form.

The invention further relates to precursors of said gas-filledmicrovesicles, in the form of a dry powder or lyophilized residue. Saidprecursor is in particular reconstitutable in the presence of a gas bycontacting it with a physiologically acceptable aqueous carrier, to forman aqueous suspension of said gas-filled microvesicles upon agitation ofthe mixture.

A further aspect of the invention relates to a pharmaceutical kitcomprising a precursor of said gas-filled microvesicles and aphysiologically acceptable aqueous carrier.

DETAILED DESCRIPTION OF THE INVENTION

The term “gas-filled microvesicles” includes any structure comprisingbubbles of gas of micrometric or nanometric size surrounded by anenvelope or layer (including film-forming layers) of a stabilizingmaterial. The term includes what is known in the art as gas-filledliposomes, microbubbles, microspheres, microballoons or microcapsules.The microvesicles are typically suspended in an aqueous carrier, inparticular a physiologically acceptable aqueous carrier. The stabilizingmaterial can be any material typically known in the art including, forinstance, surfactants, lipids, sphingolipids, oligolipids,phospholipids, proteins, polypeptides, carbohydrates, and synthetic ornatural polymeric materials.

The term “microbubbles” includes bubbles of gas suspended in an aqueouscarrier, which are bound at the gas/liquid interface by a very thinenvelope (film) involving a stabilizing amphiphilic material disposed atthe gas to liquid interface (sometimes referred to in the art as an“evanescent” envelope). Microbubble suspensions can be prepared bycontacting a suitable precursor thereof, such as powdered amphiphilicmaterials (e.g. freeze-dried preformed liposomes or freeze-dried orspray-dried phospholipid dispersions or solutions) with air or other gasand then with an aqueous carrier, while agitating to generate amicrobubble suspension which can then be administered, preferablyshortly after its preparation. Examples of aqueous suspensions of gasmicrobubbles, of precursors and of the preparation thereof aredisclosed, for instance, in U.S. Pat. No. 5,271,928, U.S. Pat. No.5,445,813, U.S. Pat. No. 5,413,774, U.S. Pat. Nos. 5,556,610, 5,597,549,U.S. Pat. No. 5,827,504 and WO 04/069284, which are here incorporated byreference in their entirety.

The terms “microballoons” or “microcapsules” include suspensions inwhich the bubbles of gas are surrounded by a solid material envelope ofa lipid or of natural or synthetic polymers. Examples of microballoonsand of the preparation thereof are disclosed, for instance, in U.S. Pat.No. 5,711,933 and U.S. Pat. No. 6,333,021.

The term polypeptide as used herein includes sequences of amino acids,which can be either synthetic or preferably natural amino acids.

The term “targeting ligand” includes any compound, moiety or residuehaving, or being capable of promoting a targeting activity towardstissues and/or receptors in vivo. Targets with which a targeting ligandmay be associated include tissues such as, for instance, myocardialtissue (including myocardial cells and cardiomyocytes), membranoustissues (including endothelium and epithelium), laminae, connectivetissue (including interstitial tissue) or tumors; blood clots; andreceptors such as, for instance, cell-surface receptors for peptidehormones, neurotransmitters, antigens, complement fragments andimmunoglobulins. The term includes in particular polypeptides comprisingamino acid sequences which exhibit binding affinity (“active sequences”)for selectins, particularly for P-selectin; said active sequencesinclude for instance amino acids 5-16, 1-19, 5-41 and 1-47 as set forthin SEQ ID NO: 1.

The term “targeted gas-filled microvesicle” includes any gas-filledmicrovesicle comprising at least one targeting ligand in itsformulation.

The phrase “intermediate of a targeted gas-filled microvesicle” includesany gas-filled microvesicle which can be converted into a targetedgas-filled microvesicle. Such intermediate may include, for instance,gas-filled microvesicles (or precursors thereof) including a suitablereactive moiety (e.g. maleimide), which can be reacted with acorresponding complementary reactive (e.g. thiol) linked to a targetingligand.

The expression “Fc region” indicates the crystallizable fragment of animmunoglobulin (Ig) composed of the carboxy-terminal halves of bothheavy chains linked to each other by disulfide bonds. Fc fragments aredifferent for each immunoglobulin class (i.e. IgG, IgM, IgA, etc.) andtype (IgG₁, IgG₂ etc.).

The term “full length Fc domain” indicates a domain composed of twoheavy chains that comprise two or three constant domains depending onthe class of the antibody. By binding to specific proteins the Fc domainensures that each antibody generates an appropriate immune response fora given antigen. This Fc domain also binds to various cell receptors,such as Fc receptors, and other immune molecules, such as complementproteins. By doing this, it mediates different physiological effectsincluding opsonization, cell lysis, and degranulation of mast cells,basophils and eosinophils. Full length Fc domain sequences compriseeither unmodified (original) amino acid sequences or corresponding aminoacid sequences where one or more non essential amino acids have beenmutated. For instance, amino acid sequence 49 to 272 of SEQ ID NO:4corresponds to the full length Fc domain of IgG₁ immunoglobulin, withthe two exceptions of amino acids 59 and 62 (where Leu and Gly residuesof the original Fc sequence have both been replaced by Ala residues).

The term “therapeutic agent” includes within its meaning any compound,moiety or residue which can be used in any therapeutic application, suchas in methods for the treatment of a disease in a patient, as well asany substance which is capable of exerting or responsible to exert abiological effect in vitro and/or in vivo. Therapeutic agents thusinclude any compound or material capable of being used in the treatment(including prevention, alleviation, pain relief or cure) of anypathological status in a patient (including malady, affliction, disease,lesion or injury). Examples of therapeutic agents are drugs,pharmaceuticals, bioactive agents, cytotoxic agents, chemotherapyagents, radiotherapeutic agents, proteins, natural or syntheticpeptides, including oligopeptides and polypeptides, vitamins, steroidsand genetic material, including nucleosides, nucleotides,oligonucleotides, polynucleotides and plasmids.

The expression “physiologically acceptable aqueous carrier” includesliquid carriers which are generally employed for injections, such as,for instance, water, typically sterile, pyrogen free water (to preventas much as possible contamination in the intermediate lyophilizedproduct), aqueous solutions such as saline (which may advantageously bebalanced so that the final product for injection is not hypotonic), oraqueous solutions of one or more tonicity adjusting substances such assalts or sugars, sugar alcohols, glycols or other non-ionic polyolmaterials (eg. glucose, sucrose, sorbitol, mannitol, glycerol,polyethylene glycols, propylene glycols and the like).

Gas-Filled Microvesicles

According to an embodiment of the present invention, the gas-filledmicrovesicles associated with a targeting ligand as above defined aremicrobubbles.

Components suitable for forming a stabilizing envelope of microbubblescomprise, for instance, phospholipids; lysophospholipids; fatty acids,such as palmitic acid, stearic acid, arachidonic acid or oleic acid;lipids bearing polymers, such as chitin, hyaluronic acid,polyvinylpyrrolidone or polyethylene glycol (PEG), also referred as“pegylated lipids”; lipids bearing sulfonated mono- di-, oligo- orpolysaccharides; cholesterol, cholesterol sulfate or cholesterolhemisuccinate; tocopherol hemisuccinate; lipids with ether orester-linked fatty acids; polymerized lipids; diacetyl phosphate;dicetyl phosphate; ceramides; polyoxyethylene fatty acid esters (such aspolyoxyethylene fatty acid stearates), polyoxyethylene fatty alcohols,polyoxyethylene fatty alcohol ethers, polyoxyethylated sorbitan fattyacid esters, glycerol polyethylene glycol ricinoleate, ethoxylatedsoybean sterols, ethoxylated castor oil or ethylene oxide (EO) andpropylene oxide (PO) block copolymers; sterol aliphatic acid estersincluding, cholesterol butyrate, cholesterol iso-butyrate, cholesterolpalmitate, cholesterol stearate, lanosterol acetate, ergosterolpalmitate, or phytosterol n-butyrate; sterol esters of sugar acidsincluding cholesterol glucuronides, lanosterol glucoronides,7-dehydrocholesterol glucoronide, ergosterol glucoronide, cholesterolgluconate, lanosterol gluconate, or ergosterol gluconate; esters ofsugar acids and alcohols including lauryl glucoronide, stearoylglucoronide, myristoyl glucoronide, lauryl gluconate, myristoylgluconate, or stearoyl gluconate; esters of sugars with aliphatic acidsincluding sucrose laurate, fructose laurate, sucrose palmitate, sucrosestearate, glucuronic acid, gluconic acid or polyuronic acid; saponinsincluding sarsasapogenin, smilagenin, hederagenin, oleanolic acid, ordigitoxigenin; glycerol or glycerol esters including glyceroltripalmitate, glycerol distearate, glycerol tristearate, glyceroldimyristate, glycerol trimyristate, glycerol dilaurate, glyceroltrilaurate, glycerol dipalmitate; long chain alcohols including n-decylalcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, or n-octadecylalcohol; 6-(5-cholesten-3β-yloxy)-1-thio-β-D-galactopyranoside;digalactosyldiglyceride;6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxy-1-thio-β-D-galactopyranoside;6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxy)-1-thio-β-D-mannopyranoside;12-(((7′-diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoicacid;N-[12-(((7′-diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoyl]-2-aminopalmiticacid; N-succinyldioleylphosphatidylethanolamine;1,2-dioleyl-sn-glycerol; 1,2-dipalmitoyl-sn-3-succinylglycerol;1,3-dipalmitoyl-2-succinylglycerol;1-hexadecyl-2-palmitoylglycerophosphoethanolamine orpalmitoylhomocysteine; alkylamines or alkylammonium salts, comprising atleast one (C₁₀-C₂₀), preferably (C₁₄-C₁₈), alkyl chain, such as, forinstance, N-stearylamine, N,N′-distearylamine, N-hexadecylamine,N,N′-dihexadecylamine, N-stearylammonium chloride,N,N′-distearylammonium chloride, N-hexadecylammonium chloride,N,N′-dihexadecylammonium chloride, dimethyldioctadecylammonium bromide(DDAB), hexadecyltrimethylammonium bromide (CTAB); tertiary orquaternary ammonium salts comprising one or preferably two (C₁₀-C₂₀),preferably (C₁₄-C₁₈), acyl chain linked to the N-atom through a (C₃-C₆)alkylene bridge, such as, for instance,1,2-distearoyl-3-trimethylammonium-propane (DSTAP),1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP),1,2-oleoyl-3-trimethylammonium-propane (DOTAP),1,2-distearoyl-3-dimethylammonium-propane (DSDAP); and mixtures orcombinations thereof.

Depending on the combination of components and on the manufacturingprocess of the microbubbles, the above listed exemplary compounds may beemployed as the main compound for forming the microbubble's envelope oras simple additives, thus being present only in minor amounts.

According to a preferred embodiment, at least one of the compoundsforming the microbubbles' envelope is an amphiphilic compound (i.e. anorganic molecule comprising both a hydrophilic and lipophilic moiety),preferably a phospholipid, optionally in admixture with any of the otherabove-cited materials. According to the present description, the termphospholipid is intended to encompass any amphiphilic phospholipidcompound, the molecules of which are capable of forming a stabilizingfilm of material (typically in the form of a mono-molecular layer) atthe gas-water boundary interface in the final microbubbles suspension.Accordingly, these materials are also referred to in the art as“film-forming phospholipids”.

Amphiphilic phospholipid compounds typically contain at least onephosphate group and at least one, preferably two, lipophilic long-chainhydrocarbon groups.

Examples of suitable phospholipids include esters of glycerol with oneor preferably two (equal or different) residues of fatty acids and withphosphoric acid, wherein the phosphoric acid residue is in turn bound toa hydrophilic group, such as, for instance, choline(phosphatidylcholines—PC), serine (phosphatidylserines—PS), glycerol(phosphatidylglycerols—PG), ethanolamine (phosphatidylethanolamines—PE),inositol (phosphatidylinositol—PI). Esters of phospholipids with onlyone residue of fatty acid are generally referred to in the art as the“lyso” forms of the phospholipid or “lysophospholipids”. Fatty acidsresidues present in the phospholipids are in general long chainaliphatic acids, typically containing from 12 to 24 carbon atoms,preferably from 14 to 22; the aliphatic chain may contain one or moreunsaturations or is preferably completely saturated. Examples ofsuitable fatty acids included in the phospholipids are, for instance,lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid,behenic acid, oleic acid, linoleic acid, and linolenic acid. Preferably,saturated fatty acids such as myristic acid, palmitic acid, stearic acidand arachidic acid are employed.

Further examples of phospholipids are phosphatidic acids, i.e. thediesters of glycerol-phosphoric acid with fatty acids; sphingolipidssuch as sphingomyelins, i.e. those phosphatidylcholine analogs where theresidue of glycerol diester with fatty acids is replaced by a ceramidechain; cardiolipins, i.e. the esters of 1,3-diphosphatidylglycerol witha fatty acid; glycolipids such as gangliosides GM1 (or GM2) orcerebrosides; glucolipids; sulfatides and glycosphingolipids.

As used herein, the term phospholipids include either naturallyoccurring, semisynthetic or synthetically prepared products that can beemployed either singularly or as mixtures.

Examples of naturally occurring phospholipids are natural lecithins(phosphatidylcholine (PC) derivatives) such as, typically, soya bean oregg yolk lecithins.

Examples of semisynthetic phospholipids are the partially or fullyhydrogenated derivatives of the naturally occurring lecithins. Preferredphospholipids are fatty acid di-esters of phosphatidylcholine,ethylphosphatidylcholine, phosphatidylglycerol, phosphatidic acid,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol or ofsphingomyelin.

Examples of preferred phospholipids are, for instance,dilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidylcholine(DMPC), dipalmitoyl-phosphatidylcholine (DPPC),diarachidoyl-phosphatidylcholine (DAPC), distearoyl-phosphatidylcholine(DSPC), dioleoyl-phosphatidylcholine (DOPC), 1,2Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC),dipentadecanoyl-phosphatidylcholine (DPDPC),1-myristoyl-2-palmitoyl-phosphatidylcholine (MPPC),1-palmitoyl-2-myristoyl-phosphatidylcholine (PMPC),1-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC),1-stearoyl-2-palmitoyl-phosphatidylcholine (SPPC),1-palmitoyl-2-oleylphosphatidylcholine (POPC),1-oleyl-2-palmitoyl-phosphatidylcholine (OPPC),dilauroyl-phosphatidylglycerol (DLPG) and its alkali metal salts,diarachidoylphosphatidyl-glycerol (DAPG) and its alkali metal salts,dimyristoylphosphatidylglycerol (DMPG) and its alkali metal salts,dipalmitoylphosphatidylglycerol (DPPG) and its alkali metal salts,distearoylphosphatidylglycerol (DSPG) and its alkali metal salts,dioleoyl-phosphatidylglycerol (DOPG) and its alkali metal salts,dimyristoyl phosphatidic acid (DMPA) and its alkali metal salts,dipalmitoyl phosphatidic acid (DPPA) and its alkali metal salts,distearoyl phosphatidic acid (DSPA), diarachidoylphosphatidic acid(DAPA) and its alkali metal salts, dimyristoyl-phosphatidylethanolamine(DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidyl-ethanolamine (DSPE), dioleylphosphatidyl-ethanolamine(DOPE), diarachidoylphosphatidyl-ethanolamine (DAPE),dilinoleylphosphatidylethanolamine (DLPE), dimyristoylphosphatidylserine (DMPS), diarachidoyl phosphatidylserine (DAPS),dipalmitoyl phosphatidylserine (DPPS), distearoylphosphatidylserine(DSPS), dioleoylphosphatidylserine (DOPS), dipalmitoyl sphingomyelin(DPSP), and distearoylsphingomyelin (DSSP),dilauroyl-phosphatidylinositol (DLPI), diarachidoylphosphatidylinositol(DAPI), dimyristoylphosphatidylinositol (DMPI),dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol(DSPI), dioleoyl-phosphatidylinositol (DOPI).

Suitable phospholipids further include phospholipids modified by linkinga hydrophilic polymer, such as polyethyleneglycol (PEG) orpolypropyleneglycol (PPG), thereto. Preferred polymer-modifiedphospholipids include “pegylated phospholipids”, i.e. phospholipidsbound to a PEG polymer. Examples of pegylated phospholipids arepegylated phosphatidylethanolamines (“PE-PEGS” in brief) i.e.phosphatidylethanolamines where the hydrophilic ethanolamine moiety islinked to a PEG molecule of variable molecular weight (e.g. from 300 to20000 daltons, preferably from 500 to 5000 daltons), such as DPPE-PEG(or DSPE-PEG, DMPE-PEG, DAPE-PEG or DOPE-PEG). For example, DPPE-PEG2000refers to DPPE having attached thereto a PEG polymer having a meanaverage molecular weight of about 2000.

Particularly preferred phospholipids are DAPC, DSPC, DSPG, DPPA, DSPA,DMPS, DPPS, DSPS and Ethyl-DSPC. Most preferred are DSPG or DSPC.

Mixtures of phospholipids can also be used, such as, for instance,mixtures of DSPE, DPPE, DPPC, DSPC and/or DAPC with DSPS, DPPS, DSPA,DPPA, DSPG, DPPG, Ethyl-DSPC and/or Ethyl-DPPC.

In preferred embodiments, the phospholipid is the main component of thestabilizing envelope of microbubbles, amounting to at least 50% (w/w) ofthe total amount of components forming the envelope of the gas-filledmicrobubbles. In some of the preferred embodiments, substantially thetotality of the envelope (i.e. at least 80% and up to 100% by weight)can be formed of phospholipids.

The phospholipids can conveniently be used in admixture with any of theabove listed compounds. Thus, for instance, substances such ascholesterol, ergosterol, phytosterol, sitosterol, lanosterol,tocopherol, propyl gallate or ascorbyl palmitate, fatty acids such asmyristic acid, palmitic acid, stearic acid, arachidic acid andderivatives thereof or butylated hydroxytoluene and/or othernon-phospholipid compounds can optionally be added to one or more of theforegoing phospholipids in proportions ranging from zero to 50% byweight, preferably up to 25%. Particularly preferred are amphiphiliccompounds, such as C₁₀-C₂₀ carboxylic acids, preferably palmitic acid.

According to a preferred embodiment, the envelope of microbubblesaccording to the invention includes a compound bearing an overall(positive or negative) net charge. Said compound can be a chargedamphiphilic material, preferably a lipid or a phospholipid.

Examples of phospholipids bearing an overall negative charge arederivatives, in particular fatty acid di-ester derivatives, ofphosphatidylserine, such as DMPS, DPPS, DSPS; of phosphatidic acid, suchas DMPA, DPPA, DSPA; of phosphatidylglycerol such as DMPG, DPPG and DSPGor of phosphatidylinositol, such as DMPI, DPPI or DPPI. Also modifiedphospholipids, in particular PEG-modified phosphatidylethanolamines,such as DPPE-PEG or DSPE-PEG, can be used as negatively chargedmolecules. Also the lyso-form of the above cited phospholipids, such aslysophosphatidylserine derivatives (e.g. lyso-DMPS, -DPPS or -DSPS),lysophosphatidic acid derivatives (e.g. lyso-DMPA, -DPPA or -DSPA) andlysophosphatidylglycerol derivatives (e.g. lyso-DMPG, -DPPG or -DSPG),can advantageously be used as negatively charged compounds. Otherexamples of negatively charged compounds are bile acid salts such ascholic acid salts, deoxycholic acid salts or glycocholic acid salts; and(C₁₂-C₂₄), preferably (C₁₄-C₂₂) fatty acid salts such as, for instance,palmitic acid salts, stearic acid salts,1,2-dipalmitoyl-sn-3-succinylglycerol salts or1,3-dipalmitoyl-2-succinylglycerol salts.

Preferably, the negatively charged compound is selected among DPPA,DPPS, DSPG, DPPG, DSPE-PEG2000, DSPE-PEG5000 or mixtures thereof.

The negatively charged component is typically associated with acorresponding positive counter-ion, which can be mono- (e.g. an alkalimetal or ammonium), di- (e.g. an alkaline earth metal) or tri-valent(e.g. aluminium). Preferably the counter-ion is selected among alkalimetal cations, such as Na⁺ or K⁺, more preferably Na⁺.

Examples of phospholipids bearing an overall positive charge arederivatives of ethylphosphatidylcholine, in particular di-esters ofethylphosphatidylcholine with fatty acids, such as1,2-distearoyl-sn-glycero-3-ethylphosphocholine (Ethyl-DSPC or DSEPC),1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (Ethyl-DPPC or DPEPC).The negative counterion is preferably a halide ion, in particularchloride or bromide ion. Examples of positively charged compounds thatcan be incorporated into the envelope of microbubbles are mono-, di-tri-, or tetra-alkylammonium salts with a halide counter ion (e.g.chloride or bromide) comprising at least one (C₁₀-C₂₀), preferably(C₁₄-C₁₈), alkyl chain, such as, for instance mono- ordi-stearylammonium chloride, mono or di-hexadecylammonium chloride,dimethyldioctadecylammonium bromide (DDAB) or hexadecyltrimethylammoniumbromide (CTAB). Further examples of positively charged compounds thatcan be incorporated into the envelope of microbubbles are tertiary orquaternary ammonium salts with a halide counter ion (e.g. chloride orbromide) comprising one or preferably two (C₁₀-C₂₀), preferably(C₁₄-C₁₈), acyl chains linked to the N-atom through a (C₃-C₆) alkylenebridge, such as, for instance,1,2-distearoyl-3-trimethylammonium-propane (DSTAP),1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP),1,2-oleoyl-3-trimethylammonium-propane (DOTAP) or1,2-distearoyl-3-dimethylammonium-propane (DSDAP).

DSEPC, DPEPC and/or DSTAP are preferably employed as positively chargedcompounds in the microbubble envelope.

The positively charged component is typically associated with acorresponding negative counter-ion, which can be mono- (e.g. halide),di- (e.g. sulphate) or tri-valent (e.g. phosphate). Preferably thecounter-ion is selected from among the halide ions, such as F⁻(fluorine), Cl⁻ (chlorine) or Br⁻ (bromine).

Mixtures of neutral and charged compounds, in particular ofphospholipids and/or lipids, can be satisfactorily employed to form themicrobubble envelope. The amount of charged lipid or phospholipid mayvary from about 95 mol % to about 0.1 mol %, with respect to the totalamount of lipid and phospholipid, preferably from 80 mol % to 0.5 mol %.

Preferred mixtures of neutral phospholipids and charged lipids orphospholipids are, for instance, DPPG/DSPC, DSTAP/DAPC, DPPS/DSPC,DPPS/DAPC, DPPE/DPPG, DSPA/DAPC, DSPA/DSPC and DSPG/DSPC.

Any of the above illustrated components useful for forming thestabilizing envelope of the gas-filled microvesicle, in particularphospholipids, preferably pegylated phospholipids, can be modified byinserting a suitable reactive moiety therein, in order to allow bindingsuitable compounds, such as a targeting ligand comprising the sequenceof amino acids set forth as SEQ ID NO:1. For instance, a pegylatedphospholipid (e.g. DSPE-PEG2000) may comprise a terminal reactive moiety(e.g. maleimide, in brief “mal”, thus forming a DSPE-PEG-mal component)capable of (covalently) reacting with a corresponding reactive moiety ona compound comprising the above sequence. Examples of additionalsuitable reactive moieties are illustrated in the following of thisspecification.

According to an alternative embodiment, the targeting ligand componentcan be associated with gas-filled microcapsules. Preferred examples ofmicrocapsules are those having a stabilizing envelope comprising apolymer, preferably a biodegradable polymer, or a biodegradablewater-insoluble lipid (such as tripalmitine) optionally in admixturewith a biodegradable polymer. Examples of suitable microcapsules and ofthe preparation thereof are disclosed, for instance in U.S. Pat. No.5,711,933 and U.S. Pat. No. 6,333,021, herein incorporated by referencein their entirety. Microcapsules having a proteinaceous envelope, i.e.made of natural proteins (albumin, haemoglobin) such as those describedin U.S. Pat. No. 4,276,885 or EP-A-0 324 938 (here incorporated byreference), can also be employed. The targeting ligand can beincorporated into the microcapsules e.g. by binding it to anenvelope-forming component of the microcapsules, according to thepreparation methods illustrated above, or by admixing to the componentsforming the microcapsules envelope an amphiphilic component, as thosepreviously illustrated, covalently bound to targeting ligand.

Other excipients or additives may be present either in the dryformulation of the microvesicles or may be added together with theaqueous carrier used for the reconstitution thereof, without necessarilybeing involved (or only partially involved) in the formation of thestabilizing envelope of the microvesicles. These include pH regulators(such as histidine), osmolality adjusters, viscosity enhancers,emulsifiers, bulking agents, etc. and may be used in conventionalamounts. For instance compounds like polyoxypropylene glycol andpolyoxyethylene glycol as well as copolymers thereof can be used.Examples of viscosity enhancers or stabilizers are compounds selectedfrom linear and cross-linked poly- and oligo-saccharides, sugars andhydrophilic polymers such as polyethylene glycol.

As the preparation of gas-filled microvesicles may involve a freezedrying or spray drying step, it may be advantageous to include in theformulation a lyophilization additive, such as an agent withcryoprotective and/or lyoprotective effect and/or a bulking agent, forexample an amino-acid such as glycine; a carbohydrate, e.g. a sugar suchas sucrose, mannitol, maltose, trehalose, glucose, lactose or acyclodextrin, or a polysaccharide such as dextran; or apolyoxyalkyleneglycol such as polyethylene glycol. Typically, the amountof the lyophilization additive may range from about 10 to about 1000times (w/w) the amount of the microvesicle-forming components.

Any biocompatible gas, gas precursor or mixture thereof may be employedto fill the above microvesicles (hereinafter also identified as“microvesicle-forming gas”).

The gas may comprise, for example, air; nitrogen; oxygen; carbondioxide; hydrogen; nitrous oxide; a noble or inert gas such as helium,argon, xenon or krypton; a radioactive gas such as Xe¹³³ or Kr⁸¹; ahyperpolarized noble gas such as hyperpolarized helium, hyperpolarizedxenon or hyperpolarized neon; a low molecular weight hydrocarbon (e.g.containing up to 7 carbon atoms), for example an alkane such as methane,ethane, propane, butane, isobutane, pentane or isopentane, a cycloalkanesuch as cyclobutane or cyclopentane, an alkene such as propene, buteneor isobutene, or an alkyne such as acetylene; an ether; a ketone; anester; halogenated gases, preferably fluorinated gases, such as orhalogenated, fluorinated or perfluorinated low molecular weighthydrocarbons (e.g. containing up to 7 carbon atoms); or a mixture of anyof the foregoing. Where a halogenated hydrocarbon is used, preferably atleast some, more preferably all, of the halogen atoms in said compoundare fluorine atoms.

Fluorinated gases are preferred, in particular perfluorinated gases,especially in the field of ultrasound imaging. Fluorinated gases includematerials which contain at least one fluorine atom such as, for instancefluorinated hydrocarbons (organic compounds containing one or morecarbon atoms and fluorine); sulfur hexafluoride; fluorinated, preferablyperfluorinated, ketones such as perfluoroacetone; and fluorinated,preferably perfluorinated, ethers such as perfluorodiethyl ether.Preferred compounds are perfluorinated gases, such as SF₆ orperfluorocarbons (perfluorinated hydrocarbons), i.e. hydrocarbons whereall the hydrogen atoms are replaced by fluorine atoms, which are knownto form particularly stable microbubble suspensions, as disclosed, forinstance, in EP 0554 213, which is herein incorporated by reference.

The term perfluorocarbon includes saturated, unsaturated, and cyclicperfluorocarbons. Examples of biocompatible, physiologically acceptableperfluorocarbons are: perfluoroalkanes, such as perfluoromethane,perfluoroethane, perfluoropropanes, perfluorobutanes (e.g.perfluoro-n-butane, optionally in admixture with other isomers such asperfluoro-isobutane), perfluoropentanes, perfluorohexanes orperfluoroheptanes; perfluoroalkenes, such as perfluoropropene,perfluorobutenes (e.g. perfluorobut-2ene) or perfluorobutadiene;perfluoroalkynes (e.g. perfluorobut-2-yne); and perfluorocycloalkanes(e.g. perfluorocyclobutane, perfluoromethylcyclobutane,perfluorodimethylcyclobutanes, perfluorotrimethylcyclobutanes,perfluorocyclopentane, perfluoromethylcyclopentane,perfluorodimethylcyclopentanes, perfluorocyclohexane,perfluoromethylcyclohexane and perfluorocycloheptane). Preferredsaturated perfluorocarbons include, for example, CF₄, C₂F₆, C₃F₈, C₄F₈,C₄F₁₀, C₅F₁₂ and C₆F₁₂.

It may also be advantageous to use a mixture of any of the above gasesin any ratio. For instance, the mixture may comprise a conventional gas,such as nitrogen, air or carbon dioxide and a gas forming a stablemicrobubble suspension, such as sulfur hexafluoride or a perfluorocarbonas indicated above. Examples of suitable gas mixtures can be found, forinstance, in WO 94/09829, which is herein incorporated by reference. Thefollowing combinations are particularly preferred: a mixture of gases(A) and (B) in which the gas (B) is a fluorinated gas, selected amongthose previously illustrated, including mixtures thereof, and (A) isselected from air, oxygen, nitrogen, carbon dioxide or mixtures thereof.The amount of gas (B) can represent from about 0.5% to about 95% v/v ofthe total mixture, preferably from about 5% to 80%.

Particularly preferred gases are SF₆, C₃F₈, C₄F₁₀ or mixtures thereof,optionally in admixture with air, oxygen, nitrogen, carbon dioxide ormixtures thereof.

In certain circumstances it may be desirable to include a precursor to agaseous substance (i.e. a material that is capable of being converted toa gas in vivo). Preferably the gaseous precursor and the gas derivedtherefrom are physiologically acceptable. The gaseous precursor may bepH-activated, photo-activated, temperature activated, etc. For example,certain perfluorocarbons may be used as temperature activated gaseousprecursors. These perfluorocarbons, such as perfluoropentane orperfluorohexane, have a liquid/gas phase transition temperature aboveroom temperature (or the temperature at which the agents are producedand/or stored) but below body temperature; thus, they undergo aliquid/gas phase transition and are converted to a gas within the humanbody.

For the use in MRI the microvesicles will preferably contain ahyperpolarized noble gas such as hyperpolarized neon, hyperpolarizedhelium, hyperpolarized xenon, or mixtures thereof, optionally inadmixture with air, carbon dioxide, oxygen, nitrogen, helium, xenon, orany of the halogenated hydrocarbons as defined above.

For use in scintigraphy, the microvesicle will preferably containradioactive gases such as Xe¹³³ or Kr⁸¹ or mixtures thereof, optionallyin admixture with air, carbon dioxide, oxygen, nitrogen, helium, kriptonor any of the halogenated hydrocarbons as defined above.

Targeting Ligand

The polypeptide associated with a microvesicle according to the presentinvention comprises at least a portion of the sequence of amino acids asset forth in SEQ ID NO: 1 which exhibits binding affinity for selectins,particularly for P-selectin. In particular, said polypeptide comprisesat least amino acids 5-16 of SEQ ID NO: 1, corresponding to amino acids46-57 of the “P-selectin glycoprotein ligand-1” (PSGL-1, GenBank Acc. NoQ14242.1). According to a preferred embodiment, the targeting ligandcomprises at least amino acids 1-19, more preferably at least aminoacids 5-41 and even more preferably at least amino acids 1-47 as setforth in SEQ ID NO: 1 (these latters corresponding to amino acids 42-88of the “P-selectin glycoprotein ligand-1”).

“P-selectin glycoprotein ligand-1” and polypeptides comprising theactive sequences illustrated above, including SEQ ID NO: 1, preferablycomprise a glycan residue bound to at least one amino acid of saidsequences.

The term “glycan residue” comprises O-linked glycan residues (linked tothe oxygen atom of hydroxyl groups of amino acid residues such asserine, threonine, tyrosine, hydroxytyrosine or hydroxyproline) andN-linked glycan residues (linked to the nitrogen atom of tertiary aminogroups of amino acids, such as asparagine).

O-linked glycans typically comprise sugar residues such asN-acetylgalactosamine, N-acetylglucosamine (GlcNAc), fucose, glucose,mannose (Man), hexose, xylose, sialic acid or mixtures thereof. O-linkedglycans preferably consist of a sialyl Lewis x structure (sLe^(x),sialic acid-galactopyranosyl-fucose-N-acetylglucosamine).

N-linked glycans typically comprise a pentasaccharidic core(Man3GlcNAc2). Complex type chains may present a mono-, bi-, tri- (2,4and 2,6 branched), tetra-, and pentaantennary structures. They mayfurther comprise various saccharides such as, galactose, sialic acid,N-acetylglucosamine, mannose, fucose and combinations of thereof.

The polypeptide associated with a microvesicle of the present inventionpreferably contains one or more of the following glycans (or mixturesthereof) bound to an amino acid of the sequence:

wherein:

-   R is either a bond or represents any other glycan as above defined;    NeuAc is neuraminic acid; Gal is galactose; GlcNAc is    N-acetylglucosamine and Fuc is fucose.

Amino acids residues of SEQ ID NO:1 which may optionally bear a glycanresidue as above defined comprise: (a) amino acids in position 16, 25,26, 28, 29, 32, 36, 39, 40 or 41, preferably bearing O-linked glycanresidues; and/or (b) amino acid in position 24 preferably bearingN-linked glycan residues.

Advantageously, SEQ ID NO: 1 may comprise a sialyl Lewis-x (sLe^(x))bound to threonine at the position 16 (see e.g. R. D Cumming, “Structureand function of selectin ligand PSGL-1”, Braz. J. Biol. Res., 32(5)1999, pp. 520-528).

Furthermore, at least one tyrosine residue in position 5, 7 and/or 10 ofSEQ ID NO: 1 may optionally be sulfated (TyrSO₃). More preferably atleast two and even more preferably all of the three tyrosine residuesmay contain a sulfate group.

The polypeptide is an amino acid sequence of from 12 to 200, morepreferably of 12 to 100, amino acid residues in length and it comprisesa reactive moiety capable of reacting with a corresponding reactivemoiety of a microvesicle's component. Particularly preferred is an aminoacid sequence of from 12 to 75 amino acid residues, even more preferablyof from 12 to 50 residues.

According to a preferred embodiment the polypeptide comprises an aminoacid bearing a reactive moiety. Said amino acid is preferably selectedfrom the group consisting of: Cysteine and/or a basic amino acid,preferably Lysine. Preferably, said reactive moiety is at the C-terminalposition of the polypeptide, preferably it being Lysine. In a preferredembodiment, the presence of a single reactive moiety (Lysine inparticular) at the C-terminus of the polypeptide sequence allows for acontrolled functionalization of said group and for subsequent effectivereaction condition with a corresponding reactive group on a component ofthe microvesicle. On the other side, the presence of a plurality ofreactive Lysine groups on a peptidic chain (such as in the case of theFc portion of SEQ ID NO:4) may render the functionalization of saidgroups much more random, with the result of having a much lower controlon which among the various reactive groups of the peptide will bind tothe microvesicles' component.

Preferred polypeptides are those represented by formula (I) and morepreferably formula (II), as previously illustrated.

In a preferred embodiment, the polypeptide comprises the amino acids ofSEQ ID NO: 1 linked to amino acid sequence SEQ ID NO:2 or to aN-terminal fragment thereof, where said fragment corresponds to aminoacids 1-20, 1-15, 1-10, 1-5 or 1-4 of SEQ ID NO:2.

When the N-terminal fragment is a single amino acid residue, said aminoacid is preferably a proline (Pro).

A particularly preferred embodiment is represented by the fusionpolypeptide having SEQ ID NO:3 and consisting of SEQ ID NO:1 covalentlybound to SEQ ID NO:2.

The fusion polypeptide can be obtained by enzymatic proteolysis ofrPSGL-Ig, for instance by incubating the protein in the presence of asuitable endoproteinase. In a preferred embodiment an endoproteasecleaving peptidyl bonds on the C-terminal side of Lysine residues (e.g.endoproteinase Lys-C) is used. The incubation of the protein withendoproteinase Lys-C allows in particular the removal of a substantialportion of the Fc domain from the mature protein rPSGL-Ig, providing adimeric sequence containing a single (C-terminal) Lysine residue (aminoacid in position 71 in SEQ ID NO: 3 and SEQ ID NO: 4) for each sequenceof the dimer, which can advantageously be employed for the subsequentbinding procedure of the polypeptide to a suitable microvesicle'scomponent.

Alternatively, the polypeptide can be obtained by production ofrecombinant protein from a custom made DNA. Briefly, a plasmid isprepared by inserting the cDNA sequence of the desired polypeptide in aplasmid vector. The plasmid vector is then associated with a suitableexpression system for producing the recombinant polypeptide (e.g. as setforth in SEQ ID NO:3). Examples of suitable expression systems includemammalian cells, such as CHO cells (Chinese Hamster Ovary cells), HEK293cells (Human Embryonic Kidney 293 cells), Sp2/0 cells (mouse myelomacell line), MEL cells (mouse erythroleukemia cells) or COS cells (kidneycells of the monkey carrying the SV40 genetic material); insect cells,such as Sf9 cell lines; virus containing cells such as BEVS cells(Baculovirus Expression Vector System); or plant-based expressionsystems, such as tobacco leaves, corn, rice cell or transgenic potatoes.Most preferably, CHO cells stably expressing core-2β1,6N-acetylglucosaminyltransferase (C2GlcNAcT-I) andα-1,3-fucosyltransferase-VII (fucT-VII) are transfected with the cDNAencoding for the polypeptide. Cells expressing permanently therecombinant polypeptide are selected. Then, these cells are transferredto a bioreactor to allow large scale production of the polypeptide.

In a preferred embodiment the recombinant polypeptide is obtained indimeric form, in particular in homodimeric form. In general, peptidedimerization is a natural process occurring in the cells expressing therecombinant polypeptide, typically during Posttranslational modification(PTM) of the polypeptide.

The above recombinant preparation technique can be used for preparing apolypeptide according to the invention, which comprises any activesequence amino acid exhibiting binding affinity for selectins,particularly p-selectin, as set forth before (including the polypeptideas set forth in SEQ ID NO:3), preferably in dimeric form. In aparticularly preferred embodiment, the method can be used for preparinga polypeptide containing a single Lysine amino acid (preferably interminal position, particularly in C-terminal position) and comprisingany of the above illustrated active sequences.

To provide the preferred dimeric form to be associated with themicrovesicle, the polypeptide preferably comprises in its sequence oneor more cysteine residues which are bound to one or more respectivecysteine residues on a corresponding polypeptide sequence, so to form atleast one (preferably at least two) disulfide bridge between the twosequences.

In an alternative embodiment, the polypeptide associated with themicrovesicle can be employed in monomeric form. Said monomeric form canbe obtained according to conventional procedures, either by directpreparation of the monomeric form or by reduction of disulfide bonds indimeric polypeptides. For instance, a suitable monomeric form oftargeting ligand can be obtained by reducing the disulfide bonds ofcysteine residues of a dimeric form, e.g. the sequence set forth as SEQID NO:3. The disulfide bond reduction can be performed according toconventional techniques, e.g. by incubating a suspension of the dimericform in the presence of suitable reducing agents, such as TCEP. Thereduction of the disulfide bond has the further advantage of providing asuitable reactive group (—SH) in the ligand, for the subsequent bindingto a corresponding moiety on a component of the microvesicle (e.g.PE-PEG-maleimide), without need of introducing a (thiolated) reactivegroup in the polypeptide.

In an embodiment of the invention, the polypeptide can be bound to thecomponent of the gas-filled microvesicle through a linker. Suitablelinkers are preferably hydrophilic residues, typically containingrepeating oxyethylene units in the backbone chain.

According to an embodiment, the linker is a moiety of formula (III):—X—(CH₂)_(f)—[O—(CH₂)_(g)]_(h)—[O—(CH₂)_(j)]_(k)—Y—  (III)

where

f, g, h and j independently represent an integer of from 1 to 4, krepresents and integer of from 0 to 4, and X and Y respectivelyrepresent respective reactive moieties for binding the linker to thepolypeptide, at one end, and to the microvesicle's component, at theother end.

The linker comprises suitable reactive moieties at its respective ends,for covalently binding to a corresponding complementary reactive moietyon the microvesicle's component, on one side, and to a correspondingcomplementary reactive moiety on the polypeptide, for instance on the Yresidue of the polypeptide of formula (I), on the other side.

Examples of said reactive moieties include amino groups (—NH₂, formingthe —NH— binding residue), carboxyl groups (—COON, forming the —CO—binding residue) or thiol groups (—SH, forming the —S— binding residue).Preferably said binding moiety is an amino or a carboxyl group.

Preferred examples of linkers of formula III are:

—CO—CH₂—[O—(CH₂)₂]₂—NH-(Adoa)

—CO—CH₂—[O—(CH₂)₂]₂—CO-(Tuda)

—NH—CH₂—(CH₂—O—CH₂)₃—CH₂—NH-(Ttda)

—CO—CH₂—[O—(CH₂)₂]₂—CO—NH—CH₂—(CH₂—O—CH₂)₃—CH₂—NH-(Ddhh)

Preferably, said linker is formed by two, equal or different, moietiesdefined by the above formula.

Examples of combined linkers are:

-Adoa-Adoa- or -Ddhh- (which is comprised of the Ttda- andTuda-linkers).

Polysaccharides, containing suitable reactive binding moieties, arefurther examples of suitable linkers.

The sequence comprising SEQ ID NO:1, or active fragments thereof, andthe desired linker can be prepared according to conventional peptidesynthesis methods.

The polypeptide as above illustrated can be associated with amicrovesicle according to any of the procedures known in the art,including for instance, covalent binding, non-covalent interactions ofaffinity binding pairs (e.g. interaction between avidin or streptavidinon one side and biotin on the other side), electrostatic interactions(e.g. ionic or hydrogen bond) or hydrophobic interactions (e.g. betweenlipophilic hydrocarbon chains).

Preferably, the polypeptide is covalently bound to a respectivecomponent of the gas-filled microvesicle.

For instance, if the polypeptide includes a reactive amino group (e.g. aprimary amino group of Lysine), it can be reacted with themicrovesicle's component containing a suitable corresponding reactivemoiety, such as an isothiocyanate group (to form a thiourea bond), areactive ester (to form an amide bond), or an aldehyde group (to form animine bond, which may be reduced to an alkylamine bond).

Alternatively, when the targeting ligand includes a reactive thiolgroup, suitable complementary reactive moieties on the microvesicle'scomponent may include haloacetyl derivatives, maleimides (to form athioether bond) or a mixed disulfide comprising a sulphide in the formof a 2-pyridylthio (PDT) group (which, upon reaction with a thiolderived from the targeting ligand, results in the formation of a stabledisulfide bond).

Alternatively, according to an embodiment of the invention, a targetingligand containing an amino reactive moiety (e.g. a secondary aminogroup, in particular the terminal —NH₂ group) can be first reacted witha sulphur-containing compound, to introduce a reactive thiol moiety inthe targeting ligand, which is then reacted with a correspondingcomplementary moiety on the microvesicle's component as aboveillustrated. Examples of suitable sulphur-containing compounds usefulfor introducing a reactive thiol moiety in a targeting ligand containinga reactive amino moiety include, for instance: thioimidate (such asTraut's reagent) N-succinimidyl-S-acetylthioacetate (SATA),N-succinimidyl-S-acetylthiopropionate (SATP) or N-succinimidyl3-(2-pyridyldithio)propionate (SPDP). Detailed description ofS-containing agents and respective thiolation reactions can be found,for instance, in the book by Greg T. Hermanson: “BioconjugateTechniques”, Elsevier ed., 2^(nd) ed. (April 2008), chapter 1, section4-1. For instance, one may prepare a maleimide-derivatized phospholipid(e.g. phosphatidylethanolamine—PE—or pegylated PE) and react it with atargeting ligand (e.g. SEQ ID NO:3) where a secondary amino group (e.g.the —NH₂ of terminal Lysine) has been previously reacted with asulphur-containing compound (such as those previously illustrated), tointroduce a reactive thiol moiety; the obtained compound can then beused in the preparation of targeted gas-filled microvesicles.

According to a further alternative, when the targeting ligand includes areactive carboxylic group, suitable reactive moieties on themicrovesicle's component can be amines and hydrazides (to form amide orN-acyl, N′-alkylhydrazide functions).

According to a preferred embodiment, a targeting ligand containing anamino reactive moiety (e.g. on a Lysine residue), can be first reactedwith a maleimide-containing compound, to introduce a reactive maleimidemoiety in the targeting ligand, which is then reacted with acorresponding complementary moiety on the microvesicle's component.Maleimide-containing agents useful for introducing a reactive maleimidemoiety in a targeting ligand containing a reactive amino moiety andrespective reaction of addition of maleimide group are well known in theart. Examples of suitable maleimide-containing compounds include, forinstance: AMAS (N-(α-maleimidoacetoxy)succinimide ester), BMPS(N-(β-maleimidopropoxyl)succinimide ester), EMCS(N-(ε-maleimidocaproyloxy)succinimide ester), GMBS(N-(γ-maleimidobutyryloxy)succinimide ester), LC-SMCC(succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate)),MBS (m-maleimidobenzoyl-N-hydroxysuccimide ester), SMCC(succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), SMPB(succinimidyl-4-(p-maleimidophenyl)butyrate), SM(PEG)n reagent(succinimidyl-(N-maleimidopropionamido)-ethyleneglycol) ester), SMPH(succinimidyl-6-((β-maleimidopropionamido)hexanoate)), sulfo-EMCS(N-(ε-maleimidocaproyloxy)sulfosuccinimide ester), sulfo-GMBS(N-(γ-maleimidobutyroyloxy)sulfosuccinimide ester), sulfo-KMUS(N-(κ-maleimidoundecanoyloxy)-sulfosuccinimide ester), sulfo-MBS(m-maleimidobenzoyl-N-hydroxysulfo-succinimide ester), sulfo-SMCC(sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate),sulfo-SMPB (sulfosuccinimidyl 4-(p-maleimidophenyl(butyrate)). Accordingto a particularly preferred embodiment, one may react a thiol-containingphospholipid (e.g. thiolated phosphatidylethanolamine—PE—or pegylatedPE) with a targeting ligand (e.g. SEQ ID NO:3) where a secondary aminogroup (e.g. the NH₂ of terminal lysine) has been previously reacted witha maleimide-containing compound (such a those previously illustrated),to introduce a reactive maleimide moiety therein; the obtained compoundcan then be used in the preparation of the microvesicles. Thethiol-containing phospholipid can be obtained, for instance, by reactinga 2-pyridyldithio (PDT) group attached to a phospholipid with areductive agent (such as TCEP (tris(2-carboxyethyl)phosphinehydrochloride) to generate a reactive thiol moiety on the phospholipid.Examples of thiol-containing phospholipids include Sodium1,2-Dipalmitoyl-sn-Glycero-3-Phosphothioethanol, (from Avanti PolarLipids, IUPAC: sodium(R)-2,3-bis(palmitoyloxy)propyl(2-mercaptoethyl)phosphonate), or thoseobtainable by chemical reduction of a respective pyridylthio-precursors,such as: sodium(R)-2,3-bis(palmitoyloxy)propyl(2-(3-mercaptopropanamido)ethyl)phosphate(from sodium1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate],Avanti Polar Lipids), sodium(R)-2,3-bis(oleoyloxy)propyl(2-(3-mercaptopropanamido)ethyl)phosphate(from sodium1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate],Avanti polar Lipids) or ammonium(R)-2,3-bis(stearoyloxy)propyl(2-(((2-(3-mercaptopropanamido)polyethyleneglycol 2000)carbonyl)amino)ethyl)phosphate (from1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethyleneglycol)-2000], Avanti Polar Lipids).

In an alternative embodiment, a biotin residue can be introduced on thepolypeptide (e.g. by reacting hydrosuccinimidobiotin with the peptide'sC-terminal) and the biotinylated peptide is then reacted with amicrovesicle comprising a streptavidin-bearing (or avidin-, neutravidin-or extravidi-bearing) component, such as a pegylated phospholipidcontaining a streptavidin (or avidin, neutravidin or extravidin)residue.

Targeted Gas-Filled Microvesicles

The targeted microvesicles of a composition according to the inventioncan be produced according to any known method in the art, as illustratedin the above cited patent documents.

For instance, the manufacturing method of microbubbles may involve thepreparation of a dried powdered material comprising an amphiphilicmaterial as indicated above, preferably by lyophilization (freezedrying) of an aqueous and/or organic suspension/emulsion comprising saidmaterial. Said dried powdered material, identified in the presentspecification and claims as “precursor” of the gas-filled microvesicles,is then contacted with a physiologically acceptable solution in thepresence of the desired gas, to form the desired suspension ofgas-filled microvesicles upon agitation of the mixture.

According to the preparation method described in WO 91/15244,film-forming amphiphilic compounds can be first converted into alamellar form by any method employed for formation of liposomes. To thisend, an aqueous solution comprising the film forming lipids andoptionally other additives (e.g. viscosity enhancers, non-film formingsurfactants, electrolytes etc.) can be submitted to high-speedmechanical homogenisation or to sonication under acoustic or ultrasonicfrequencies, and then freeze dried to form a free flowing powder whichis then stored in the presence of a gas. Optional washing steps, asdisclosed for instance in U.S. Pat. No. 5,597,549, can be performedbefore freeze drying.

According to an alternative embodiment (described for instance in U.S.Pat. No. 5,597,549) a film forming compound and a hydrophilic stabiliser(e.g. polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol,glycolic acid, malic acid or maltol) can be dissolved in an organicsolvent (e.g. tertiary butanol, 2-methyl-2-butanol or C₂Cl₄F₂) and thesolution can be freeze-dried to form a dry powder.

Preferably, as disclosed for instance in International patentapplication WO2004/069284, a phospholipid (selected among those citedabove and including at least one of the above-identified chargedphospholipids) and a lyoprotecting agent (such as those previouslylisted, in particular carbohydrates, sugar alcohols, polyglycols,polyoxyalkylene glycols and mixtures thereof) can be dispersed in anemulsion of water with a water immiscible organic solvent (e.g. branchedor linear alkanes, alkenes, cyclo-alkanes, aromatic hydrocarbons, alkylethers, ketones, halogenated hydrocarbons, perfluorinated hydrocarbonsor mixtures thereof) under agitation. The emulsion can be obtained bysubmitting the aqueous medium and the solvent in the presence of atleast one phospholipid to any appropriate emulsion-generating techniqueknown in the art. Preferably, the phospholipid is dispersed in theaqueous medium before this latter is admixed with the organic solvent.Alternatively, the phospholipid can be dispersed in the organic solventor it may be separately added the aqueous-organic mixture before orduring the emulsification step. The so obtained microemulsion, whichcontains microdroplets of solvent surrounded and stabilized by thephospholipid material (and optionally by other amphiphilic film-formingcompounds and/or additives), is then lyophilized according toconventional techniques to obtain a lyophilized material, which isstored (e.g. in a vial in the presence of a suitable gas) and which canbe reconstituted with an aqueous carrier to finally give a gas-filledmicrobubbles suspension where the dimensions and size distribution ofthe microbubbles are substantially comparable with the dimensions andsize distribution of the suspension of microdroplets.

A further process for preparing gas-filled microbubbles comprisesgenerating a gas microbubble dispersion by submitting an aqueous mediumcomprising a phospholipid (and optionally other amphiphilic film-formingcompounds and/or additives) to a controlled high agitation energy (e.g.by means of a rotor stator mixer or by sonication) in the presence of adesired gas and using the obtained mixture as such or subjecting theobtained dispersion to lyophilisation to yield a dried reconstitutableproduct. An example of this process is given, for instance, inWO97/29782, here enclosed by reference.

Spray drying techniques (as disclosed for instance in U.S. Pat. No.5,605,673) can also be used to obtain a dried powder, reconstitutableupon contact with physiological aqueous carrier to obtain gas-filledmicrobubbles.

The precursor in dried or lyophilized form obtained with any of theabove techniques will generally be in the form of a powder or a cake,and can be stored (e.g. in a vial) in contact with the desired gas. Theprecursor is readily reconstitutable, in the presence of the desiredgas, in a suitable physiologically acceptable aqueous liquid carrier,which is typically injectable, to form the gas-filled microbubbles, upongentle agitation of the suspension. Suitable physiologically acceptableliquid carriers are sterile water, aqueous solutions such as saline(which may advantageously be balanced so that the final product forinjection is not hypotonic), or solutions of one or more tonicityadjusting substances such as salts or sugars, sugar alcohols, glycols orother non-ionic polyol materials (eg. glucose, sucrose, sorbitol,mannitol, glycerol, polyethylene glycols, propylene glycols and thelike).

According to an embodiment of the invention, a targeting construct (i.e.comprising the targeting ligand bound to a component of themicrovesicle) can be admixed as such with the other components of theformulation, so to be incorporated into the stabilizing envelope uponreconstitution of the freeze-dried material obtained according to any ofthe above preparation methods.

Alternatively, the initial formulation of microbubbles may containsuitably intermediate functionalized component (e.g. amaleimide-containing phosphatidylethanolamine), to produce afreeze-dried material containing said intermediate; the targetingligand, containing a suitable complementary reactive moiety (e.g.thiol), is then linked, by reacting the respective reactive moieties, tothe intermediate functionalized compound already incorporated in theenvelope of the reconstituted microbubbles.

In the case of the process disclosed in WO2004/069284, the targetingconstruct comprising the targeting ligand bound to the microvesicle'scomponent can also be admixed with the components of the initialmixture, undergoing to the emulsion and lyophilisation steps.Alternatively, a micellar suspension containing the targeting constructcan be separately prepared and subsequently added to the already formedemulsion (containing the other film-forming components), preferablyunder heating. As above, instead of the formed construct, afunctionalized intermediate can alternatively be used, which can then bereacted at any step of the process (e.g. in the emulsion phase or uponreconstitution of the lyophilized compound) with a targeting ligandcontaining a complementary reactive moiety. According to an embodiment,a functionalized envelope-forming component (or envelope-forming/spacerintermediate construct) is added as a micellar suspension to the formedemulsion, under agitation. The targeting ligand (containing thecomplementary reactive moiety) is then added to the obtained emulsion.

According to a preferred embodiment the peptide sequence as set forth inSEQ ID NO:3, in dimeric form, is first reacted with a thiolating agent(selected, for instance, among those previously illustrated) tointroduce a reactive thiol group on the primary amino group of theC-terminal Lysine residue. The thiolating agent is preferably employedin a molar excess with respect to the Lysine residue, preferably fromabout 5 to 200 times molar excess, more preferably from 20 to 100 timesand even more preferably of about 50 times. The thiolated peptide isthen added to a suspension of a maleimide-containing component ofgas-filled microvesicles (e.g. a maleimide-modified pegylatedphospholipid, such as DSPE-PEG-maleimide). The mixture is then incubatedand the obtained construct (comprising SEQ ID NO:3 and themicrovesicle's component) can be used for the subsequent preparationsteps of the gas-filled microvesicles, as above illustrated.

The amount of targeting ligand bound to the surface of a microvesicle isselected so as to preferably provide a multivalent microvesicle, i.e. amicrovesicle comprising a plurality of targeting ligand on its surface.In general, the microvesicle comprises at least 200 targeting moleculesper μm² of microvesicles surface, preferably at least 500 molecules/μm²,more preferably at least 1000 molecules/μm², and even more preferably atleast 2000 molecules/μm². On the other side, as a too high concentrationof targeting ligand on the surface of the microvesicle is notnecessarily required, the microvesicle generally comprises less than15000 targeting molecules per μm² of microvesicle surface, preferablyless than 12000 molecules/μm², more preferably less than 10000molecules/μm², and even more preferably less than 8000 molecules/μm².

The amount of targeting ligand bound at the surface of a microvesiclecan be determined according to common techniques known in the art. Forinstance, the total surface of the envelope of the microvesicles in amicrovesicles suspension can be first determined, e.g. by CoulterCounter measurement. Then, the total amount of molecules of targetingligand in the microvesicles suspension can be determined, e.g. bymeasuring the total amount of a chemical marker of the targeting ligand(for instance sialic acid or a specific amino acid), for instance byLiquid-Chromatography Mass Spectrometry (LC-MS). The density oftargeting ligand on the microvesicles surface can then be easilycalculated.

Use of Targeted Microvesicles

The targeted gas-filled microvesicles of the invention can be used inany in vitro or in vivo analysis requiring the detection of receptorscapable of binding to the targeting ligand above identified, such astissues or cells expressing a selectin receptor, preferably E-selectinand/or P-selectin receptors, more preferably p-selectin receptors. Inparticular the microvesicles of the invention are useful in diagnosticmethods for detecting possible pathological conditions of vascularendothelium, in particular in connection with inflammatory processes(e.g. acute coronary syndrome, angiogenesis, rheumatoid arthritis,Crohn's disease, etc.) and, more in general, of any organ or tissueexpressing P-selectin and/or E-selectin. Furthermore, microvesiclesaccording to the invention can be employed as an efficient diagnostictool during the (therapeutic) treatment of a patient suffering from aninflammatory disease or pathology, where “during” includes any timebefore the beginning of the treatment, in the course of said treatmentand/or at the end of said treatment. For instance the microvesicles ofthe invention can advantageously be employed in the monitoring and/orfollow-up of an anti-inflammatory treatment (e.g. of any of the abovecited diseases or pathologies), e.g. to determine or evaluate theeffects of the administration of an anti-inflammatory orinflammatory-inhibitor drug on the disease or pathology. In a preferredembodiment, during a treatment a region of interest of the patient issubjected to ultrasound imaging upon administration of the microvesiclesof the invention, for instance at regular time intervals, at apredetermined time interval after each drug administration ortherapeutic intervention and/or after a selected number of drugadministrations or treatments; a final imaging of the region of interestis then preferably performed at the end or conclusion of the treatment.

The gas-filled microvesicles of the invention can further be used intherapeutic-associated imaging methods, said therapeutic-associatedimaging including any method for the treatment of a disease in a patientwhich comprises the use of a contrast imaging agent (e.g. for thedelivery of a therapeutic compound to a selected receptor or tissue),and which is capable of exerting or is responsible to exert a biologicaleffect in vitro and/or in vivo. Therapeutic-associated imaging mayadvantageously be associated with the controlled localized destructionof the gas-filled microvesicles, e.g. by means of ultrasound waves athigh acoustic pressure (typically higher than the one generally employedin non-destructive diagnostic imaging methods). This controlleddestruction may be used, for instance, for the treatment of blood clots(a technique also known as sonothrombolysis), optionally in combinationwith the release of a suitable therapeutic compound associated with thecontrast agent. Alternatively, said therapeutic-associated imaging mayinclude the delivery of a therapeutic agent into cells, as a result of atransient membrane permeabilization at the cellular level induced by thelocalized burst or activation of the microvesicles. This technique canbe used, for instance, for an effective delivery of genetic materialinto the cells; alternatively, a drug can be locally delivered,optionally in combination with genetic material, thus allowing acombined pharmaceutical/genetic therapy of the patient (e.g. in case oftumor treatment). The therapeutic agent can be associated with thegas-filled microvesicle according to conventional methods, or can beadministered as a separate compound of the composition.

Typically, an effective amount of the contrast agent is administered(e.g. by injection) to a patient in need thereof and the body part ortissue of the patient to be imaged or treated (“region of interest”) issubjected to the desired imaging method. Preferably, the contrast agentis administered intravenously. The term patient includes any subject(human or animal) undergoing the administration of the contrast agent,either for diagnostic/therapeutic purposes or for experimental purposes(including, for instance, use of a contrast agent in laboratory animals,e.g. to follow an experimental therapeutic treatment).

According to a preferred embodiment, an effective amount of targetedmicrovesicles is administered to a patient, typically by injection of asuspension thereof. The imaging of the region of interest will thus beenhanced by the presence of the microvesicles bound to the receptor inthe region of interest.

A variety of imaging techniques may be employed in ultrasoundapplications, for example including fundamental and non-linear (e.g.harmonic) B-mode imaging, pulse or phase inversion imaging andfundamental and non-linear Doppler imaging; if desired three- orfour-dimensional imaging techniques may be used. Furthermore, diagnostictechniques entailing the destruction of gas-filled microvesicles (e.g.by means of ultrasound waves at high acoustical pressure) which arehighly sensitive detection methods are also contemplated.

Microvesicles according to the invention can typically be administeredin a concentration of from about 0.01 to about 5.0 μl of gas (entrappedinside the microvesicles) per kg of patient, depending e.g. on theirrespective composition, the tissue or organ to be imaged and/or thechosen imaging technique. This general concentration range can of coursevary depending from specific imaging applications, e.g. when signals canbe observed at very low doses such as in color Doppler or power pulseinversion. Possible other diagnostic imaging applications includescintigraphy, optical imaging, photo-acoustic imaging, magneticresonance imaging and X-ray imaging, including X-ray phase contrastimaging.

The following examples will help to further illustrate the invention.

EXAMPLES

The following materials and abbreviations have been used in thefollowing examples.

-   -   DSPC Distearoylphosphatidylcholine (Genzyme)    -   Palmitic acid Palmitic acid, Hexadecanoic acid (Fluke)    -   DSPE-PEG2000 Distearoylphosphatidylethanolamine modified with        PEG2000, sodium salt (Genzyme)    -   DSPE-PEG2000-mal Distearoylphosphatidylethanolamine modified        with PEG2000-maleimide (Avanti Polar lipids)    -   DSPE-PEG2000-PDP 1,2        Distearoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate        (polyethylene glycol)-2000] ammonium salt (Avanti Polar Lipids)    -   PDP Pyridyldithiopropionyl    -   Traut reagent 2-Iminothiolane hydrochloride (Pierce)    -   SATA N-Succinimidyl S-Acetylthioacetate (Pierce)    -   Sulfo-SMCC (sulfosuccinimidyl        4-[N-maleimidomethyl]cyclohexane-1-carboxylate) (Pierce)    -   Hydroxylamine.HCl Hydroxylamine hydrochloride (Fluke)    -   EDTA.4Na Ethylenediamine tetraacetic acid, tetra sodium salt        (Fluke)    -   PEG4000 Polyglycol 4000S from Clariant    -   Cyclooctane Fluka    -   TCEP Tris(2-carboxyethyl)-phosphine hydrochloride (Pierce)    -   rPSGL-Ig Glycosylated SEQ ID NO: 4, obtained according to US        2003/0166521, Example 1    -   Lys-C Endoprotease Lys-C (Pierce, #90051)    -   Fr-1 Purified fragment of rPSGL-Ig (SEQ ID NO:3)

Example 1 Enzymatic Digestion of rPSGL-Ig for the Preparation of Fr-1

2 mg of rPSGL-Ig in 200 μL of digestion buffer (Tris.HCl 25 mM-EDTA 1mM-pH 8.5) were placed in a microcentrifuge tube, to which a solution ofLys-C (40 μg dissolved in 50 μL of distilled water) was added. The vialcontaining the powder was rinsed with 50 μL of distilled water and addedto the microcentrifuge tube. Then, the mixture was incubated 18 h at 37°C. in the Dry Block Heater.

Example 2 Separation of Fr-1 by Anion Exchange Chromatography

A chromatographic separation column (Econo-column from Bio-Rad, Vt=3.6mL, height 9.4 cm) was filled with ANX Sepharose gel (GE Healthcare) andequilibrated in a buffer containing sodium acetate 0.05 M-NaCl 0.05 M(pH 4.0). The column was run with 3-4 volumes of the starting buffer, toallow the gel to settle. The flow rate was approximately 0.23 mL/min.

290 μL of the suspension containing the digested protein obtained inExample 1 were diluted with 440 μL of acetate 0.05 M-NaCl 0.05 M-pH 4.0buffer. This mixture was applied to the column and the column was elutedwith acetate 0.05 M/NaCl buffers pH=4.0 at increasing NaClconcentrations ranging from 0.05 to 1 M.

2 mL fractions were collected during elution. Protein content in eachfraction was assessed by a OD (Optical Density) measurement at 280 nm(OD280), and presence of sugar residues were determined by theresorcinol titration. Fr-1 was recovered in fractions eluting at 1 MNaCl concentration. The fractions containing Fr-1 were collected andused for subsequent preparations. Purity of the Fr-1-containingfractions was assessed by SDS-PAGE analysis and LC-UV. The meanmolecular weight of Fr-1 (about 32 kDaltons) was determined by MALDI-ToF(Matrix-assisted laser desorption/ionization—time-of-fly).

Example 3 Thiolation of rPSGL-Ig (Comparative)

An aliquot of rPSGL-Ig stock solution (789 μL-15.1 mg of rPSGL-Ig-188.75nmoles) was diluted with 200 μL of PBE (Phosphate buffer 25 mM, 150 mMsaline, 1 mM EDTA, pH 8).

A solution of Traut reagent (2.76 mg/mL-20 mM) was prepared in PBE and75 μL of this solution were added to the rPSGL-Ig solution. Theresulting mixture was incubated at room temperature for 1 h understirring. This solution was spun through a spin-column (Zeba spin column5 mL, Pierce, #89891) equilibrated in phosphate buffer 20 mM pH 6. Thefinal volume of the solution was of about 1.2 mL (thiolated rPSGL-Igconcentration: approx. 111 nmoles/mL).

The rPSGL-Ig content in the final solution was determined by UVspectrometry at 280 nm.

The thiolated rPSGL-Ig was used immediately after purification to limitthe possible oxidation of thiol groups.

Example 4 Preparation of Microvesicles with rPSGL-Ig Ligand(Comparative)

DSPE-PEG-maleimide (6.6 mg-2.24 μmoles) was dissolved in phosphatebuffer 20 mM pH 6 (0.5 mL) at 45° C. with stirring (vortex) to obtain aclear solution. 0.5 mL of the resulting solution were then added to 59.5mL of a PEG4000 10% solution.

60 mg of a mixture of DSPC/Palmitic acid (80/20 by moles) were dissolvedin cyclooctane (4.8 mL) at 70° C.

The above prepared aqueous and organic solutions were admixed by using ahigh speed homogenizer (Megatron MT3000) for 5 min (11′500 rpm), toobtain an emulsion. The resulting emulsion was heated under stirring at60° C. for 1 h, then cooled at room temperature (about 22° C.). Theemulsion was divided in 10 mL fractions in polypropylene tubes(Falcon-15 mL).

Thiolated rPSGL-Ig prepared according to example 3 (15 nmoles) was addedto 10 mL of the emulsion and the resulting mixture was agitated at 22°C. for 2 h 30 min. The obtained emulsion was finally diluted twice with10 mL of 10% PEG4000 solution and sampled in DIN4R vials (300 μL pervial). Vials were frozen at −50° C. for 2 h (Christ Epsilonlyophilizer), then freeze-dried at −25° C. and 0.2 mbar for 12 h. Thelyophilized product was then exposed to an atmosphere containingperfluoro-n-butane and air (35/65 v/v) and the vials were sealed.

The product was dispersed in a volume of saline (1 mL, 150 mM NaCl) bygentle hand shaking before use.

Example 5 Thiolation of Fr-1

Dried Fr-1 (17.1 nmoles) from example 2 was dissolved in 160 μL of PBE(Phosphate buffer 25 mM, 150 mM saline, 1 mM EDTA, pH 8). A solution ofSATA 10 mg/mL was prepared in anhydrous DMSO and 4 μL (10 equivalents ofSATA) of this solution was added in the Fr-1 solution. The obtainedsolution was incubated for 30 min at room temperature. The solution wasthen diluted with PBE (150 μL). This solution was spun through aspin-column (Zeba spin column 2 mL, Pierce, #89890) equilibrated in PBE(using 50 μL of PBE as stacker). The final volume of the solution was ofabout 360 μL.

A solution of hydroxylamine hydrochloride (0.696 g) and EDTA.tetrasodiumsalt (0.19 g) was prepared in PBE (15 mL). The pH of this solution wasadjusted to 7.3 with NaOH 10 N and the volume was completed to 20 mL. Aaliquot of this deacetylation solution (40 μL) was added to the Fr-1solution (360 μL). The obtained solution was incubated for 2 h at roomtemperature. This solution was spun through a spin-column (Zeba spincolumn 2 mL, Pierce, #89890) equilibrated in phosphate buffer 20 mM pH 6(using 50 μL of PBE as stacker). The final volume of the solution was ofabout 450 μL (thiolated Fr-1 concentration: approx. 33 nmoles/mL).

The thiolated Fr-1 was used immediately after purification to limit thepossible oxidation of thiol groups.

Example 6 Preparation of Microvesicles with Fr-1

DSPE-PEG-maleimide (6.6 mg-2.24 μmoles) was dissolved in phosphatebuffer 20 mM pH 6 (0.5 mL) at 45° C. with stirring (vortex) to obtain aclear solution. 0.5 mL of the resulting solution were then added to 59.5mL of PEG4000 10% solution.

60 mg of a mixture of DSPC/Palmitic acid (80/20 by moles) were dissolvedin cyclooctane (4.8 mL) at 70° C.

The above prepared aqueous and organic solutions were admixed by using ahigh speed homogenizer (Megatron MT3000) for 5 min (11′500 rpm), toobtain an emulsion. The resulting emulsion was heated under stirring at60° C. for 1 h, then cooled at room temperature (about 22° C.). Theemulsion was divided in 10 mL fractions in PP tubes (Falcon-15 mL).

Fr-1 (prepared according example 5, 13 nmoles) was added to 10 mL of theemulsion and the resulting mixture was gently stirred at 22° C. for 2 h30 min. The obtained emulsion was finally diluted twice with 10% PEG4000solution and sampled in DIN4R vials (300 μL per vial). Vials were frozenat −50° C. for 2 h (Christ Epsilon lyophilizer), then freeze-dried at−25° C. and 0.2 mbar for 12 h. The lyophilized product was then exposedto an atmosphere containing perfluoro-n-butane and air (35/65 v/v) andthe vials were sealed.

The product was dispersed in a volume of saline (1 mL, 150 mM NaCl) bygentle hand shaking before use.

Example 7 Reduction of Fr-1 with TCEP: Monomeric Fr-1

Dried Fr-1 (fragmented and purified from 3 mg of rPSGL-Ig accordingexample 1 and example 2) was dissolved in 250 μL of buffer (Tris/HCl 50mM, 50 mM EDTA, pH 6.8).

A solution of TCEP (2.86 mg/mL-10 mM) was prepared in the same bufferand 28 μL of this solution were added to the fragment solution. Theresulting mixture was incubated at 37° C. for 1 h under stirring. Afterdilution with 100 μL of buffer, this solution was spun through aspin-column (Zeba spin column 2 mL, Pierce) equilibrated in Phosphatebuffer 20 mM pH 6. The final volume of the solution was about 0.4 mL.

A reduced monomeric Fr-1 was obtained and this compound was immediatelyused after purification (to limit the possible reoxidation of thiolgroups).

Example 8 Preparation of Microvesicles with Monomeric Fr-1 (After TCEPReduction)

Microvesicles were prepared according example 6, with the differencethat the Fr-1 solution was replaced by a solution of monomeric Fr-1 (100nmol) prepared according to example 7.

Example 9 Preparation of Fr1-SMCC

Fr-1 (76.5 nmoles) from example 2 was dissolved in 500 μL of buffer(Phosphate buffer 200 mM, 50 mM saline, 1 mM EDTA, pH 7.5). A solutionof Sulfo-SMCC 55 mg/mL was prepared in anhydrous DMSO and 62 μL (100equivalents of Sulfo-SMCC) of this solution was added to the Fr-1solution. The solution was incubated at room temperature for 45 min.This solution was spun through a spin-column (Zeba spin column 5 mL,Pierce, #89890) equilibrated in Phosphate buffer 20 mM pH 6. The finalvolume of the solution was of about 660 μL.

Example 10 Preparation of DSPE-PEG-SH

DSPE-PEG2000-PDP (4.4 mg-1473 nmoles) was dissolved in 400 μL ofPhosphate buffer (100 mM pH 6) at 40° C. with stirring (vortex) toobtain a clear solution. A 25 mM solution of TCEP in buffer (125 μL) wasadded. The obtained solution was incubated for 45 min at roomtemperature with stirring.

A sample of the solution was diluted in buffer and checked for theabsence of 2-pyridine thione.

The solution was spun through a spin-column (Zeba spin column 5 mL,Pierce, #89890) equilibrated in Phosphate buffer 20 mM pH 6. The finalvolume of the solution was of about 620 μL.

Example 11 Preparation of DSPE-PEG-SH/Fr-1-SMCC Conjugate

630 μL of Fr1-SMCC solution (70 nmoles) obtained in example 9 was addedin 470 μL of DSPE-PEG-SH solution (1050 nmoles) obtained in example 10.The solution was incubated for three hours at room temperature withstirring (rotating wheel).

The solution was then purified by anion exchange chromatography with anANX Sepharose gel (GE Healthcare).

The solution containing the purified Fr-1 conjugate (2.6 mL) was spunthrough a spin-column (Zeba spin column 10 mL, Pierce, #89893)equilibrated in Phosphate buffer 20 mM pH 6 and used for subsequentpreparations.

Example 12 Preparation of Microvesicles with DSPE-PEG-SH/Fr-1-SMCCConjugate

10 mg of a mixture of DSPC and Palmitic acid (80/20 by moles) weredissolved in cyclooctane (0.8 mL) at 70° C.

Separately, the DSPE-PEG-SH/Fr-1-SMCC conjugate solution preparedaccording to example 11 (0.75 mL-20 nmoles) was added to 9.25 mL ofPEG4000 10% solution.

The above prepared organic and aqueous solutions were admixed by using ahigh speed homogenizer (Polytron PT3000) for 1 min (8′000 rpm), toobtain an emulsion. The resulting emulsion was heated under stirring at60° C. for 1 h, then cooled at room temperature (about 22° C.).

The obtained emulsion was diluted twice with 10% PEG4000 solution andsampled in DIN4R vials (300 μL per vial). Vials were frozen at −50° C.for 2 h (Christ Epsilon lyophilizer), then freeze-dried at −25° C. and0.2 mbar for 12 h. The lyophilized product was then exposed to anatmosphere containing perfluoro-n-butane and air (35/65 v/v) and thevials were sealed.

The product was dispersed in a volume of saline (1 mL, 150 mM NaCl) bygentle hand shaking.

Example 13 Preparation of Microvesicles with DSPE-PEG-SH/Fr-1-SMCCConjugate

10 mg of a mixture of DSPC and Palmitic acid (80/20 by moles) weredispersed in distilled water (10 mL) at 70° C. for 15 min and thencooled to room temperature; the DSPE-PEG-SH/Fr-1-SMCC conjugate solutionprepared according to example 11 (20 nmoles) was then added to thedispersion under stirring.

Cyclooctane (0.8 mL) was admixed with the obtained dispersion by using ahigh speed homogenizer (Polytron PT3000) for 1 min (8′000 rpm). Theresulting emulsion was heated under stirring at 60° C. for 1 h, thencooled at room temperature (about 22° C.).

The emulsion was diluted twice with 20% PEG4000 solution and sampled inDIN4R vials (300 μL per vial). Vials were frozen at −50° C. for 2 h(Christ Epsilon lyophilizer), then freeze-dried at −25° C. and 0.2 mbarfor 12 h. The lyophilized product was then exposed to an atmospherecontaining perfluoro-n-butane and air (35/65 v/v) and the vials weresealed.

The product was dispersed in a volume of saline (1 mL, 150 mM NaCl) bygentle hand shaking.

Example 14 Preparation of Microvesicles with rPSGL-Ig Ligand(Comparative)

DSPE-PEG-mal (0.44 mg-0.15 μmole) was dissolved in phosphate buffer 20mM pH 6 (0.1 mL) at 45° C. with stirring (vortex) to obtain a clearsolution. Thiolated rPSGL-Ig prepared according example 3 (16 nmoles-144μL-0.8 nmoles/mL emulsion) was added to the solution and the resultingmixture was agitated at 22° C. for 2 h 30 min. 0.25 mL of the solutionwere then added to 19.75 mL of PEG4000 10% solution.

20 mg of a mixture of DSPC/Palmitic acid (80/20 by moles) were dissolvedin cyclooctane (1.6 mL) at 70° C.

The above prepared aqueous and organic solutions were admixed by using ahigh speed homogenizer (Polytron PT3000) for 1 min (11′000 rpm), toobtain an emulsion. The resulting emulsion was heated under stirring at60° C. for 1 h, then cooled at room temperature (about 22° C.).

The obtained emulsion was finally diluted twice with 10% PEG4000solution and sampled in DIN4R vials (300 μL per vial). Vials were frozenat −50° C. for 2 h (Christ Epsilon lyophilizer), then freeze-dried at−25° C. and 0.2 mbar for 12 h. The lyophilized product was then exposedto an atmosphere containing perfluoro-n-butane and air (35/65 v/v) andthe vials were sealed.

Example 15 Preparation of Microvesicles with Fr-1

Microvesicles were prepared according to example 14 except thatthiolated rPSGL-Ig was replaced by thiolated Fr-1 (25 nmoles, preparedaccording example 5).

Example 16 Physico-Chemical Characterization After Dispersion ofLigand-Containing Microvesicles

The freeze-dried product obtained in comparative example 14 wasdispersed by gentle shaking in a volume of saline (1 mL, 150 mM NaCl),in order to obtain an isotonic microvesicles suspension ready forintravenous injection. The microvesicles suspension was subjected tosize analysis immediately after preparation of the suspension (Time=0min), and 30 min after preparation (Time=30 min). The size distributionand concentration of microvesicles were measured with a Multisizer™ 3Coulter Counter® fitted with a 30 μm aperture tube (Dilution: 50 μL ofmicrovesicles suspension in 100 mL NaCl 0.9% solution—Analytical volume:100 μL). The preparation was characterized to determine the meandiameter in number and median diameter in volume of microvesicles (Dnand Dv50 in μm), as well as their concentration in number, wereobtained.

Similarly, also the freeze-dried product obtained in example 15 wasdispersed in an equal volume of saline and the size and distribution ofthe microvesicles in the suspension were determined as above indicated(Time=0 or 30 min).

Results are provided in the following table 1.

TABLE 1 Physico-chemical characterization of microvesicles suspensionsDiameter Diameter Microvesicle conc. Example Time [min] Dv50 [μm] Dn[μm] [×10⁸/mL] 14 (comp) 0 3.0 1.5 11.5 14 (comp) 30 2.5 1.3 18.3 15 02.7 1.3 16.8 15 30 2.6 1.3 15.8

As inferable from the above results, microvesicles with rPSGL-Ig ligandsuffered from aggregation after dispersion in saline, graduallydisaggregating over time, which is not desirable for an injectable form.On the contrary, the size, distribution and vesicles count for the Fr-1containing microvesicles were substantially constant when compared atT=0 min and at T=30 min after dispersion.

Example 17 Image Analysis of Microvesicles Suspensions AfterReconstitution in 0.9% NaCl

Microvesicles suspensions obtained according to example 14 and example15 were diluted 1/10 in 0.9% NaCl and a 10 μL aliquotes were introducedinto a Neubauer counting cell (Blaubrand®, Brand GmbH), under an opticalmicroscope (Leica Cambridge Ltd, fitted with a 20× objective lens), formicrovesicles image acquisition. The microvesicles were allowed to riseto the cover slip at the top of the Neubauer cell (2 to 3 min) and afterfocusing, images were taken with the digital camera. The images werethen analysed with a mathematical processor, to determine the amount ofunbound microvesicles, based on the assumption that a pure circularshape in the image corresponds to a single non-aggregated microvesicle,while aggregations of microvesicles produce undetected non-circularshapes. To detect “pure circular shapes” in the grayscale images, thecircular Hough transform was implemented in Matlab (The Mathworks Inc.,Natick, Mass.). The program outputs the center positions and radii ofthe detected circular shapes. The following results (Table 2) wereobserved.

TABLE 2 Image analysis by Hough transform object detection Number ofnon-aggregated microvesicles Preparation Vial #1 Vial #2 Vial #3 Example14 157 131 172 Example 15 332 354 352

As inferable from the results in Table 2, microvesicles containing theFr-1 fragment are much less prone to aggregation than microvesiclescontaining the entire protein rPSGL-Ig.

Example 18 In Vitro Binding Activity of Targeted Microvesicles

To test the effective binding, targeted microvesicles prepared accordingto comparative example 4 were injected in a flow chamber set upcomprising a coating of mouse Fc P-Selectin (CD62P-Fc Chimera, from R&DSystems (Minneapolis, Minn., USA). Microvesicles (at equivalent numberof 80×10⁶/400 μL TBS++) were injected through the flow chamber (FCS2,Bioptech, USA) in a bolus fashion and their adhesion onto the mouseP-selectin coating layer was assessed over a period of 10 min at a flowrate of 1.0 mL/min (shear rate of 714 s⁻¹) in the presence of 50% humanplasma in PBS (v:v, Biomeda collected on citrate, ref. ES1020P, Stehelin& Cie AG). A quantitative analysis of microvesicles accumulation wasperformed by counting the number of microvesicles adhering in theobserved area at 2 min intervals over the total 10 min infusion, usingthe image processing program Analysis FIVE (SIS, Germany). After 10 min,five pictures were taken randomly and the number of bound microvesicleswas measured and expressed as the number of bound bubbles at 10 min(NBB). Each observed area was 183×137 μm, as measured with the aid of astage micrometer. Measurement was performed between the middle and theexit of the chamber.

Similarly, suspensions of targeted microvesicles prepared according toexample 6 (Fr-1 targeting ligand) and according to example 8 (monomericFR-1) were injected in a flow chamber as described above, and theirbinding activity determined according to the above procedure.

Table 3 shows the results of the three tests.

TABLE 3 Number of bound microveicles at 10 min (NBM 10 min) PreparationNBM 10 min Example 4 75 ± 8 Example 6 98 ± 7 Example 8 88 ± 9

As inferable from the above results, the binding activity ofmicrovesicles containing Fr-1 is higher with respect to correspondingpreparations of microvesicles containing monomeric Fr-1 or the completeprotein rPSGL-Ig.

Example 19 In Vivo Performance of Microvesicles with Dimeric andMonomeric Fr-1

Microvesicles prepared according to examples 6 and 8, were compared inan inflammatory rat model. Inflammation was induced in the hind limb byan intramuscular injection of lipopolysaccharide (LPS, 026:B6 SigmaL-8274, 2.1 mg/kg). The effective binding of the targeted microvesicleswas evaluated by ultrasound imaging 24 h after induction of theinflammatory process. Ultrasound imaging was performed using a SiemensSequoia 512 scanner (Siemens Medical Systems, Issaquah, Wash.) equippedwith a 15L8 linear transducer (transmit frequency, 7 MHz; dynamic range,83 dB; depth, 20 mm; Time-Gain compensation (TGC): linear). 10 min aftersingle dose injections of microvesicles obtained from example 6 and fromexample 8, a quantitative analysis of microvesicles binding wasperformed using a quantification software developed in-house (BraccoSuisse S A, Geneva, Switzerland) designed to quantify contrastecho-power amplitude within areas of interest (AOI). Contrastenhancement in the AOI of the stored frames was expressed as relativeecho-power values (rms²), which are proportional to the number ofmicrovesicles in the selected AOI. Results are shown in table 4.

TABLE 4 Echo power in inflamed rat muscle Preparation Echo power 10 min(rms²) Example 6 43 ± 18 Example 8 18 ± 10As inferable from the above table, the microvesicles of example 6 (withdimeric Fr-1) result in a higher in-vivo binding with respect to themicrovesicles of example 8 (with monomeric Fr-1).

Example 20 Monitoring the Effects of Anti-Inflammatory Therapy with Fr-1Microvesicles

Microvesicles prepared according to example 6 were administered in aninflammatory rat model. Inflammation was induced in the hind limb by anintramuscular injection of lipopolysaccharide (LPS, 026:B6 Sigma L-8274,2.1 mg/kg). Monitoring of anti-inflammatory treatment efficacy wasperformed by pre-treating animals twenty four hours before LPSadministration, with a sub-cutaneous injection of etanercept (0.45mg/kg, Wyeth) or of saline. The in vivo binding activity of Fr-1microvesicles was determined according to the imaging protocol describedin example 19. The known inhibition of inflammation achieved byadministration of etanercept to prevent TNFα activity (Campbell, S. J.,Jiang, Y., Davis, A. E., Farrands, R., Holbrook, J., Leppert, D., andAnthony, D. C. (2007), Immunomodulatory effects of etanercept in a modelof brain injury act through attenuation of the acute-phase response. J.Neurochem. 103, 2245-2255) was visualized using Fr-1 microvesicles.Animals pre-treated with etanercept, showed a decrease in Fr-1microvesicles accumulation, in comparison to control animals receivingsaline. This study shows the ability of Fr-1 microvesicles to monitorexpression of selectin receptors in an inflammation site during ananti-inflammatory treatment with an inflammation inhibitor.

The invention claimed is:
 1. An aqueous suspension comprising agas-filled microvesicle, said microvesicle comprising a targetingconstruct comprising: a) an amphiphilic compound; and b) a homodimericpolypeptide consisting of two polypeptide monomers, wherein eachpolypeptide monomer consists of at most 200 amino acid residues,comprises a single lysine amino acid residue, at least amino acids 5-16as set forth in SEQ ID NO:1 and at least one cysteine residue; whereinthe peptide monomers form a disulfide bond with the respective cysteineresidues; said homodimeric polypeptide being covalently associated withsaid amphiphilic compound.
 2. The aqueous suspension according to claim1 wherein said polypeptide monomers comprise at least amino acids 1-19as set forth in SEQ ID NO:
 1. 3. The aqueous suspension according toclaim 1 wherein said polypeptide monomers comprise at least amino acids5-41 as set forth in SEQ ID NO:
 1. 4. The aqueous suspension accordingto claim 1 wherein said polypeptide monomers comprise at least aminoacids 1-47 as set forth in SEQ ID NO:
 1. 5. The aqueous suspensionaccording to claim 1 wherein said polypeptide monomers comprise at most100 amino acid residues.
 6. The aqueous suspension according to claim 1wherein said polypeptide monomers comprise at most 75 amino acidresidues.
 7. The aqueous suspension according to claim 1 wherein saidpolypeptide monomers consist of the amino acid sequence as set forth inSEQ ID NO:
 3. 8. The aqueous suspension according to claim 1 whereinsaid polypeptide monomers consist of the amino acid sequence of formula(I):(X^(A))_(n)—Y—(X^(B))_(m)  (I) where: (X^(A))_(n) represents a sequenceof n amino acids X^(A), where: n is an integer of from 12 to 199; X^(A)is any amino acid with the exclusion of lysine; and (X^(A))_(n) furthercomprises at least amino acids 5-16 as set forth in SEQ ID NO:1 and atleast one cysteine; (X^(B))_(m) represents a sequence of m amino acidsX^(B), where: m is an integer from 0 to 10, with the proviso that thesum m+n is at most 199; and X^(B) is any amino acid with the exclusionof lysine and cysteine; and Y is lysine.
 9. The aqueous suspensionaccording to claim 8 wherein (X^(A))_(n) comprises at least amino acids1-19 as set forth in SEQ ID NO:
 1. 10. The aqueous suspension accordingto claim 8 wherein (X^(A))_(n) comprises at least amino acids 5-41 asset forth in SEQ ID NO:
 1. 11. The aqueous suspension according to claim8 wherein (X^(A))_(n) comprises at least amino acids 1-47 as set forthin SEQ ID NO:
 1. 12. The aqueous suspension according to claim 8 whereinn is an integer of from 12 to 99 and m+n is at most
 99. 13. The aqueoussuspension according to claim 8 wherein n is an integer of from 12 to 74and m+n is at most
 74. 14. The aqueous suspension according to claim 8wherein X^(A) comprises two Cysteine residues.
 15. The aqueoussuspension according to any one of claim 1 or 8 wherein said polypeptidemonomers are glycosylated comprising an O-glycan moiety.
 16. The aqueoussuspension according to claim 15 wherein said O-glycan moiety comprisesa sialyl Lewis x structure.
 17. The aqueous suspension according toclaim 15 comprising one or more glycan residues bound to amino acids inpositions 16, 24, 25, 26, 28, 29, 32, 36, 39, 40 and/or 41 of SEQ IDNO:1.
 18. The aqueous suspension according to any one of claim 1 or 8wherein said polypeptide monomers further comprise a sulfate group boundto a tyrosine.
 19. The aqueous suspension according to claim 1 whereinsaid amphiphilic compound of said targeting construct is a phospholipid.20. The aqueous suspension according to any one of claim 1 or 8 whereinsaid microvesicle comprises a stabilizing amphiphilic material.
 21. Theaqueous suspension according to claim 20 wherein said amphiphilicmaterial is a phospholipid.
 22. The aqueous suspension according to anyone of claim 1 or 8 wherein the gas contained in the microvesicle is afluorinated gas, optionally in admixture with air or nitrogen.
 23. Theaqueous suspension according to claim 1 wherein said homodimericpolypeptide is covalently associated with said amphiphilic compoundthrough a lysine residue.
 24. A precursor of a gas-filled microvesicleas defined in any one of claim 1 or 8 said precursor being in driedpowdered form and comprising a microvesicle-forming material incombination with a lyophilization additive, said precursor beingreconstitutable to form an aqueous suspension comprising said gas-filledmicrovesicle upon contact and agitation with an aqueous carrier in thepresence of a gas.
 25. The precursor according to claim 24 wherein thelyophilization additive is an amino acid, a sugar, a polysaccharide orpolyaoxyalkyleneglycol.
 26. A pharmaceutical kit comprising a precursoraccording to claim 24 and a physiologically acceptable aqueous carrier,b) subjecting said patient to ultrasound imaging.
 27. The pharmaceuticalkit according to claim 26 wherein said precursor is in contact with abiocompatible gas.
 28. The pharmaceutical kit according to claim 27wherein said gas is a fluorinated gas, optionally in admixture with airor nitrogen.
 29. A method of diagnostic imaging which comprises: a)administering an effective amount of a suspension of gas-filledmicrovesicles according to any one of claim 1 or 8 to a patient; b)subjecting said patient to ultrasound imaging.
 30. The method accordingto claim 29 wherein said imaging is performed on a patient sufferingfrom an inflammatory disease or pathology during a treatment thereof.